1. Field of the Invention
The invention pertains to the field of virology. More particularly, the invention pertains to a genetically engineered baculovirus in which the native envelope protein is absent, and the virus particles are pseudotyped with an envelope protein from another virus.
2. Description of Related Art
Baculoviruses constitute a family of viruses that are pathogenic To certain insect species, but do not appear to productively infect other invertebrates or vertebrates. Baculoviruses, such as the Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV), have been developed as biological control agents and as protein expression vectors. AcMNPV also serves as the primary model system for studies of baculovirus gene regulation and structure. Baculoviruses have also been used to deliver genes into mammalian cells, and can be used as vectors for human gene therapy.
AcMNPV has a large double stranded DNA genome (134 kbp) and produces two virion phenotypes during the infection cycle. One virion phenotype, the Occlusion Derived Virus or ODV, is adapted for survival in the environment and propagation of infection from animal to animal, through oral transmission and infection of the midgut epithelial cells. In contrast, the other virion phenotype (Budded Virus or BV) is adapted for propagation of infection from cell to cell throughout the animal, after infection is established in the midgut. BV efficiently enter many cell types in the infected animal, including most notably: hemocytes, tracheal epithelial cells, and fatbody cells. The infection cycle is completed when ODV are assembled (enveloped) and occluded within occlusion bodies in the nuclei of these secondarily infected cells. Occlusion bodies are then released by cell lysis.
Because BV are generated only after successful infection of the midgut epithelial cells, BV appear to have adopted a strategy of promiscuous infection of many insect cell types. Studies of baculovirus BV entry into mammalian cell lines and cultured primary cells show that in culture, BV from AcMNPV can efficiently enter primary rat hepatocytes as well as a number of human cell lines, although baculoviruses do not productively replicate there. When a reporter gene driven by a mammalian promoter is inserted into the AcMNPV genome, expression can be readily detected in many mammalian cell types. In contrast, gene expression could not be detected from a reporter under the control of the baculovirus polyhedrin promoter. Thus, baculoviruses appear to efficiently enter mammalian cells and selectively express genes under the control of mammalian promoters. Thus, baculovirus BV can be an effective means for gene delivery to mammalian cells, as gene therapy agents.
Indeed, several features of baculoviruses are highly desirable for the development of baculoviruses as potential vectors for gene therapy. These include the capacity of the baculovirus genome to accommodate very large insertions of foreign DNA, the inability of the virus to replicate within mammalian cells, and the apparent absence of expression of most baculovirus genes. Other studies have shown that baculoviruses incorporating selectable markers (such as the neomycin phosphotransferase II gene) under a mammalian regulatory context, can be used to generate stably transformed mammalian cell lines.
The AcMNPV GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. GP64 is also required for viral assembly and efficient production of budded virions (BV) during viral exit.
During virion entry, the AcMNPV GP64 protein is involved in binding of virions to host cells. GP64 also mediates low pH triggered membrane fusion during entry by endocytosis. Genetic studies with gp64-null viruses (containing a gp64 knockout) showed that GP64 is also necessary for efficient virion budding from the cell surface. Interestingly, AcMNPV viruses containing C-terminal truncations of GP64 that removed portions or all of the cytoplasmic tail domain (CTD) did not show the same severity of the defect in budding as the complete GP64 deletion. This indicates that the CTD is not required for efficient budding and that some other feature of GP64 is important for virion assembly and budding. Although the absence of GP64 resulted in an approximately 98% reduction in virion budding, deletion of the CTD resulted in only an approximately 50% reduction in budding efficiency, indicating that other portions of the GP64 protein may play important roles in budding.
GP64 is highly conserved among a number of baculoviruses (such as AcMNPV and OpMNPV) that are relatively closely related, yet several more distantly related baculoviruses possess an unrelated envelope protein that appears to serve as a functional homolog of GP64. The major BV envelope proteins from two of these more distantly related viruses, SeMNPV (Se8) and LdMNPV (Ld130), are both envelope fusion proteins and thus serve at least one of the important functions of GP64. However, these proteins and a homolog from XcGV show a higher degree of divergence than that observed among GP64 proteins. It has therefore been proposed that GP64 may represent a more recent acquisition of an envelope glycoprotein in the Baculoviridae. Several orthomyxoviruses contain an envelope protein, GP75, that is phylogenetically related to the baculovirus GP64 protein. The GP75 proteins have been identified from only a small subset of the orthomyxoviruses and GP75 is distinct from the HA proteins found in other orthomyxoviruses. Therefore, it is possible that the GP75 protein was also recently acquired by a member of the orthomyxovirus family.
In a previous study (Barsoum et al., Hum Gene Ther. 8: 2011-8 (1997), the complete disclosure of which is hereby incorporated herein by reference), a baculovirus expressing the VSV-G protein was reported to have an enhanced ability to transduce mammalian cells. In that study, G was expressed in the presence of wt GP64, presumably generating virions with a mosaic of GP64 and G protein in the envelopes. G protein did not appear to interfere with the infectivity of the virus in insect cells but enhanced infectivity of mammalian cells. However, VSV-G protein expression in a baculovirus expression vector in the presence of wt GP64 resulted in a report of virions that were distended and sometimes contained tail-like projections. A potential problem with the utilization of baculovirus virions (BV) containing GP64 for mammalian transduction in vivo, is the rapid detection of GP64 and inactivation of the virus. The use of VSV-G protein or other suitable envelope or membrane proteins substituted for GP64 in the BV envelope could therefore provide benefits for use of baculoviruses in vivo. Retroviruses pseudotyped with VSV-G appear to be more resistant to inactivation by complement than wild type retroviruses, and this may also be true for G-pseudotyped baculovirus particles.
In other enveloped viruses, the role of the major envelope protein in virion budding is highly variable. For example, retroviruses such as HIV-1 or RSV do not require the envelope protein (Env) for virion budding, although virions generated in the absence of Env are not infectious. In contrast, envelope proteins from influenza viruses are believed to encode important functions necessary for virion budding and also influence virion morphology, and these important functions are thought to be redundant in the hemagglutinin (HA) and neuraminidase (NA) proteins of Influenza A virus. Rhabdoviruses such as VSV and Rabies viruses require the major envelope protein (G protein) for efficient budding. In the absence of G, budding of VSV or Rabies virions is reduced by approximately 97%. Heterologous proteins substituted for G can partially complement virion budding in VSV and Rabies rhabdoviruses, and studies suggest that important signals necessary for efficient budding reside in non-specific signals in the cytoplasmic tail domain. Efficient budding of VSV in the absence of intact G protein can be reconstituted by providing only a xe2x80x98stemxe2x80x99 region containing the membrane proximal portion of the G protein ectodomain (12 amino acids) combined with the transmembrane and cytoplasmic tail domains. The small xe2x80x98stemxe2x80x99 region appears to be a functional budding domain necessary to promote efficient budding of VSV in the absence of the majority of the G protein.
One hypothesis to explain the synergistic roles of various proteins in the budding process is the push-pull model, in which the push represents the role of matrix and perhaps other proteins on the inner surface of the plasma membrane, and the pull represents the role of the membrane proteins within and on the exterior of the membrane. Budding may be accomplished by the concerted or synergistic effects of the two components. While a very low level of budding may be observed in the absence of one component, efficient budding would require the activities of both components.
In certain retrovirus and rhabdovirus systems, heterologous envelope proteins can complement the absence of the endogenous envelope protein. Virions carrying a heterologous envelope protein are referred to as xe2x80x98pseudotypedxe2x80x99 viruses. Pseudotyped virions have been used for applications such as gene therapy, but also serve as valuable tools for dissecting the functions necessary for assembly of mature virions and budding at the cell surface. Thus, to better understand the requirements for baculovirus budding, we investigated whether heterologous viral glycoproteins can complement the deletion of the gp64 gene from the AcMNPV genome.
We investigated whether a heterologous viral envelope protein, the Vesicular Stomatitis Virus (VSV) G protein, can complement the deletion of GP64 in a gp64-null baculovirus, vAc64-. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9VSV-G) that inducibly expresses the VSV-G protein upon infection with AcMNPV. Sf9VSV-G and Sf9 cells were infected with vAc64- and cells were monitored for infection and for movement of infection from cell to cell. vAc64- formed plaques on Sf9VSV-G cells, but not on Sf9 cells. Plaques formed on Sf9VSV-G cells were observed only after prolonged intervals. Passage and amplification of vAc64- on Sf9VSV-G cells resulted in pseudotyped virus particles that contained the VSV-G-protein. Cell-to-cell propagation of vAc64- in the G-expressing cells is delayed in comparison to wt AcMNPV, and growth curves showed that pseudotyped vAc64- are generated at titres of approximately 106 to 107 infectious units (IU)/ml, compared with titres of approximately 108 IU/ml for wt AcMNPV in the same cells.
Propagation and amplification of pseudotyped vAc64- virions in Sf9VSV-G cells indicates that the VSV-G-protein possesses the necessary signals for baculovirus BV assembly and budding at the cell surface, or otherwise facilitates production of infectious baculovirus virions. The functional complementation of gp64-null viruses by VSV-G protein is further demonstrated by identification of a vAc64--derived virus that acquired the G gene through recombination with Sf9VSV-G cellular DNA. Gp64-null viruses expressing the VSV-G gene are capable of productive infection, replication, and propagation in Sf9 cells. We thus demonstrate herein that several functions of GP64 can be substituted by the VSV-G protein, and we provide the first example of functionally pseudotyped baculovirus virions. We further demonstrate that the VSV-G and two other heterologous envelope protein genes (i.e., two different F proteins from baculovirus Group 2 NPVs) can be engineered into the gp64-null virus genome and functionally complement the absence of GP64. Our results are consistent with a push-pull model for baculovirus budding.
The present invention shows that by expressing VSV-G or another heterologous envelope protein (whether from a cell line, or from the virus), the virus is able to propagate and to infect insect cells. Thus, the VSV-G protein and other heterologous envelope proteins can be inserted into baculovirus virions that contain no GP64 (i.e., a gp64-null baculovirus). We propose that this system is useful for generating baculoviruses targeted to specific cell types (depending on the type of protein or other molecule used to replace GP64). Also, GP64 is a target of immune recognition by the mammalian host (in vivo), and removing GP64 allows the engineered viruses to escape immune surveillance and subsequent inactivation. Immune recognition of GP64 has been demonstrated with recombinant baculoviruses in vivo (injected into mice).
In addition, gp64-null viruses can be used to great advantage in protein expression systems. A foreign gene for expression can be cloned into the GP64-null virus and the virus propagated in a complementing cell line. When the virus is used to infect normal host cells, the foreign protein is expressed, but no virus particles are budded into the culture medium. Thus, purification of expressed foreign proteins is facilitated, as contaminating virus particles do not need to be removed.
We expressed VSV-G (and two other heterologous viral envelope proteins) in the absence of GP64 and found that VSV-G complements virion infectivity and possibly virion budding, although the efficiency of infectious virion production appears to be relatively low. This represents the first example of pseudotyping of baculovirus virions in the absence of the baculovirus GP64 protein. We propose that such pseudotyped baculovirus virions are useful in potential gene therapy applications.